We have been investigating cellular signaling and its regulation using the beta-adrenergic receptor-coupled adenylyl cyclase as a model. Recently, we have focused on differences in regulation of the three beta-subtypes, B1AR, B2AR and B3AR. They are members of the G protein-coupled receptor (GPCR) superfamily and activate adenylyl cyclase via the stimulatory G protein (Gs) in response to the endogenous catecholamines epinephrine and norepinephrine. The three subtypes differ in tissue distribution with B2AR being the predominant or only subtype in most tissues; B1AR, the predominant subtype in brain and heart; and B3AR, mainly in adipocytes. They also differ in their ability to be regulated (B2AR> B1AR>>B3AR) as shown by us and others. This may provide a physiological basis for the existence of three subtypes. To compare the regulation of B1AR and B2AR, we used baby hamster kidney (BHK) and human embryo kidney (HEK 293) cells expressing similar levels of either subtype. Previously, we showed that: (i) B1AR undergoes less agonist-mediated internalization than B2AR; (ii) over-expressing GPCR kinases (GRKs) or arrestins enhances internalization of B1AR to that of B2AR; (iii) B1AR is endocytosed by the clathrin-coated pit pathway similar to that of B2AR; (iv) during or soon after endocytosis, the two subtypes are sorted to different endosomal compartments; (v) upon removal of agonist, both subtypes recycle at similar rates but recycling of B2AR and not B1AR is inhibited by the ionophore monensin which blocks endosomal acidification; and (vi) upon prolonged agonist treatment, B2AR undergoes down-regulation whereas B1AR exhibits up-regulation. As the initial step in the regulation of receptors is their phosphorylation by protein kinase A (PKA) and by GRKs, we determined the phosphorylation of the two subtypes expressed in BHK cells. The cells were labeled with P-32 phosphate and stimulated with agonist for different times. After the cells were lysed and solubilized, the labeled receptors were immunoprecipitated with anti-subtype antibodies, resolved by SDS-PAGE, and quantified by phosphor imaging. Whereas agonist stimulation increased the phosphorylation of B2AR by 3-fold over basal in 5 min, there was no increase in phosphorylation of B1AR. We performed similar experiments with cells over-expressing GRK2 and found that agonist stimulation caused a 5.7-fold increase in phosphorylation of B2AR but no increase in that of B1AR. The latter was unexpected as agonist-mediated internalization of B1AR increased from 15 to 30% with GRK2 expression. We can only speculate about the GRK2-mediated increase in B1AR internalization without an increase in phosphorylation. GRK2 has a clathrin-binding motif and can bind to the clathrin heavy chain. A B1AR-GRK2 fusion protein was shown to internalize to a greater extent than wild-type B1AR and the internalization was arrestin-independent. It is possible that at the very high levels of expression in our cells, GRK2 is acting as an adapter protein to recruit B1AR to clathrin-coated pits. Next we compared the phosphorylation of wild type and chimeric receptors in which the entire C-tail of each subtype had been exchanged for that of the other. In contrast to the wild type B1AR, the chimera B1AR with a B2AR C-tail displayed over a 2-fold increase in phosphorylation. Phosphorylation of the B2AR chimera with a B1AR C-tail was not increased by agonist stimulation. Thus the C-tails appear to be the major determinants of subtype phosphorylation as they are for internalization and down-regulation. Our current model is that the differences in agonist-stimulated phosphorylation of the two subtypes results in differences in affinity for arrestin binding. Weaker binding of arrestin to B1AR in turn leads to less internalization. Once internalized, the two subtypes diverge in their intracellular trafficking due to differences in their phosphorylation.